In mammalian cell mutagenicity assays, mutant and wild-type cells are mixed during the expression phase. This leads to a decrease in the mutant to wild-type ratio if some of the mutant progenitors or mutants grow more slowly than the wild-type cells during the expression period and the mutant fraction will not accurately represent the number of mutational events that occurred. In order to accurately quantitate the mutagenic response of the cells, we have developed an in situ procedure which segregates and immobilizes cells during expression. In these experiments, L5178Y mouse lymphoma cells heterozygous at the tk locus were plated in semisolid medium immediately after treatment and allowed to form microcolonies. The selective agent, trifluorothymidlne (TFT) was added as an overlay at specified times permitting only tk-/- cells to survive. In this procedure, each mutation is captured as an individual colony so the measured mutation frequency accurately reflects the mutational events that occur at the selected locus. In addition, the induced mutant colonies arising in the agar are the result of independent mutational events. We have found that mutation rate measured in the in situ assay was 50-fold greater than when the cells expressed the phenotype in suspension. We evaluated the effect of chemical treatment on the mutation frequency as a function of the time of TFT addition. We generated expression curves for the alkylating agents EMS and MMS and for hycanthone methanesulfonate. The numbers of TFTr colonies at various times after treatment indicated that chemically-treated cultures had higher mutation fractions than the solvent controls. For most chemicals tested, the numbers of chemically-induced mutant colonies began to increase after an expression time of 20-30 hours and began to plateau at about 50 hours. A 40 to 50 hour expression time would be recommended for use in determining chemically-induced dose responses. This approach has also been applied to AS52 cell mutation assays at the gpt gene.